Biosensor

ABSTRACT

In a biosensor for measuring a specific substance in a liquid sample, one or a combination of sugar alcohol, metallic salt, organic acid or organic acid salt which has at least one carboxyl group in a molecule, and organic acid or organic acid salt which has at least one carboxyl group and one amino group in a molecule, is included in a reagent layer provided on electrodes, thereby providing a highly-accurate biosensor which is excellent in stability and has high response (sensitivity, linearity) of the sensor to the substrate concentration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/979,842, filed May, 1, 2002, now U.S. Pat. No. 6,911,131, which is a35 U.S.C. 371 National Stage of PCT International Patent Application No.PCT/JP01/02558, filed Mar. 28, 2001, which claims priority of JapanesePatent Application No. 2000-90362, filed Mar. 29, 2000, and of JapanesePatent Application No. 2000-342537, filed Nov. 9, 2000, the contents ofall of which are hereby incorporated by reference into the subjectapplication.

TECHNICAL FIELD

The present invention relates to a biosensor for analyzing a specificcomponent in a liquid sample and, more particularly, to a reagentformulation for composing a reagent layer of the biosensor.

BACKGROUND ART

A biosensor is a sensor which utilizes the molecule identifyingabilities of organic materials such as microorganisms, enzymes, andantibodies, and applies the organic materials as molecular recognitionelements. That is, the biosensor utilizes a reaction which occurs whenan immobilized organic material recognizes a target specific substance,such as oxygen consumption by respiration of a microorganism, an enzymereaction, and luminescence.

Among biosensors, enzyme sensors have been put to practical use. Forexample, enzyme sensors for glucose, lactic acid, cholesterol, lactose,urea, and amino acid are utilized in medical measurement or foodindustry. An enzyme sensor reduces an electron acceptor by an electrongenerated by a reaction between a substrate included in a samplesolution as a specimen and an enzyme, and a measuring deviceelectrochemically measures the reduction quantity of the electronacceptor, thereby performing quantitative analysis of the specimen. Asan example of such biosensor, a sensor proposed in Japanese PublishedPatent Application No. Hei. 11-324511 has been known.

FIGS. 11 and 12 are exploded perspective views illustrating conventionalbiosensors for measuring a blood sugar level. A measuring electrode 2 or102 (also referred to as a working electrode), a counter electrode 3 or103, and a detecting electrode 4, which electrodes comprise a conductivematerial, are formed on an insulating support 1 or 101 comprisingpolyethylene terephthalate or the like, and a reagent layer 5 or 105including an enzyme which specifically reacts to a specific component ina sample solution, an electron transfer agent, and a hydrophilic polymeris formed on these electrodes.

In order to form a cavity for detecting an electric current valuegenerated by a reaction between the specific component in the samplesolution and a reagent in the reagent layer 5 or 105 with theabove-mentioned electrodes 2, 3, 4, 102, 103, a spacer 6 or 106 having aspindly cutout part 7 or 107 in a position on the electrodes and thereagent layer, and a cover 8 or 108 having an air vent 9 or 109 areattached onto the insulating support.

In the biosensor constructed as described above, the sample solution issupplied from the inlet of the cavity (sample suction inlet) to theinside of the cavity by capillary phenomenon and is let to the positionof the electrodes and the reagent layer. When a specific component inthe sample solution reacts to the reagent of the reagent layer, anelectric current is generated, and the generated electric current isread by an external measuring device through leads of the biosensor,whereby quantitative analysis of the specimen is carried out.

However, in the biosensor with the above-described reagent composition,under the environment where heat and moisture exist, particularly underthe environment of high temperature and humidity where the temperatureis over 30° C. and the humidity is over 80%, a reduction reaction occursbetween the electron transfer agent and a portion of enzyme protein orhydrophilic polymer which is included in the reagent layer 5 or 105,thereby generating a background electric current (noise electriccurrent). As the value of the background electric current increases withtime, the sensor performance is deteriorated.

Furthermore, as a means to solve the problem, it is possible toeliminate moisture and prevent the deterioration of the sensorperformance by enclosing a desiccant such as silica gel or activatedalumina into a biosensor preservation container which employs a moldedcontainer of resin or aluminium seal. However, it is impossible tocompletely eliminate water of molecular level remaining in the reagentincluded in the biosensor, with the desiccant alone.

Further, it is extremely hard to keep the preservation container free ofmoisture penetration over long term, and the reduction reaction betweena portion of enzyme protein or hydrophilic polymer and the electrontransfer agent is promoted when only a slight amount of moisture exists.Therefore, it is extremely difficult to effectively suppress theincrease in the background electric current with time.

Further, when an inorganic salt such as potassium ferricyanide isincluded in the mixed reagent layer composed of various reagents such asan enzyme and an electron transfer agent, the reagent layer is extremelyeasily crystallized in the process of drying the reagent solution,whereby the surface of the reagent layer becomes rough and uneven,resulting in deterioration in the response (linearity, sensitivity) ofthe sensor to the substrate concentration and the measurement accuracy.

The present invention is made to solve the above-mentioned problems andhas for its object to provide a highly-accurate biosensor whichefficiently prevents deterioration of the performance of the biosensordue to contact with moisture, and has high response (linearity,sensitivity) of the sensor to the substrate concentration.

DISCLOSURE OF THE INVENTION

According to Claim 1 of the present invention, there is provided abiosensor for measuring the concentration of a specific substance in asample solution, wherein sugar alcohol is included in apreviously-provided reagent layer so that it is dissolved in the samplesolution and specifically reacts to the specific substance in the samplesolution. Therefore, it is possible to suppress an increase in abackground electric current with time, and suppress a needles reactionwith various contaminants existing in the blood, without preventing anenzyme reaction or the like, thereby providing a high-performancebiosensor which is excellent in linearity (having a big slope and asmall intercept of a regression expression) and has small variationsamong individual sensors.

According to Claim 2 of the present invention, in the biosensor asdefined in Claim 1, the concentration of the specific substance ismeasured employing electrodes comprising at least a working electrodeand a counter electrode provided on an insulating support. Therefore, itis possible to provide a biosensor which is suitable for an examinationemploying electrodes.

According to Claim 3 of the present invention, in the biosensor asdefined in Claim 2, the reagent layer is formed on the electrodes or sothat the electrodes are arranged in a diffusion area where a reagent inthe reagent layer is dissolved and diffused in the sample solution, andthe reagent layer includes at least an enzyme and an electron transferagent. Therefore, it is possible to provide a biosensor which issuitable for an examination employing a reaction between an enzyme andan electron transfer agent.

According to Claim 4 of the present invention, in the biosensor asdefined in any of Claims 1 to 3, the sugar alcohol is chain polyhydricalcohol or cyclic sugar alcohol, or a substitution product or derivativeof the sugar alcohol. Claim 4 embodies the sugar alcohol defined inClaim 1 and, therefore, the same effect as in Claim 1 can be achieved.

According to Claim 5 of the present invention, in the biosensor asdefined in any of Claims 1 to 3, the sugar alcohol is either or both ofmultiol and lactitol. Claim 5 embodies the sugar alcohol in Claim 1 andtherefore, the same effects as in Claim 1 are achieved.

According to Claim 6 of the present invention, there is provided abiosensor for measuring the concentration of a specific substance in asample solution, wherein metallic salt is included in apreviously-provided reagent layer so that it is dissolved in the samplesolution and specifically reacts to the specific substance in the samplesolution. Therefore, it is possible to provide a biosensor whichsuppresses an increase in a background electric current with time,without preventing an enzyme reaction or the like.

According to Claim 7 of the present invention, in the biosensor asdefined in Claim 6, the concentration of the specific substance ismeasured employing electrodes comprising at least a working electrodeand a counter electrode provided on an insulating support. Therefore, itis possible to provide a biosensor which is suitable for an examinationemploying electrodes.

According to Claim 8 of the present invention, in the biosensor asdefined in Claim 7, the reagent layer is formed on the electrodes or sothat the electrodes are arranged in a diffusion area where a reagent inthe reagent layer is dissolved and diffused in the sample solution, andthe reagent layer includes at least an enzyme and an electron transferagent. Therefore, it is possible to provide a biosensor which issuitable for an examination employing a reaction between an enzyme andan electron transfer agent.

According to Claim 9 of the present invention, in the biosensor asdefined in any of Claims 6 to 8, the metallic salt is metallic saltsulfate, metallic salt hydrogensulfate, metallic salt sulfite, metallicsalt hydrogensulfite, or metallic salt hyposulfite. Claim 9 embodies themetallic salt in Claim 6 and, therefore, the same effect as in Claim 6is achieved.

According to Claim 10 of the present invention, in the biosensor asdefined in any of Claims 6 to 8, the metallic salt is either or both ofmagnesium sulfate and calcium sulfate. Claim 10 embodies the metallicsalt in Claim 6 and, therefore, the same effect as in Claim 6 isachieved.

According to Claim 11 of the present invention, in the biosensor asdefined in any of Claims 6 to 8, the metallic salt is metallic saltnitrate, metallic salt hydrogennitrate, metallic salt nitrite, metallicsalt hydrogennitrite, or metallic salt hyponitrite. Claim 11 embodiesthe metallic salt in Claim 6 and, therefore, the same effect as in Claim6 is achieved.

According to Claim 12 of the present invention, in the biosensor asdefined in any of Claims 6 to 8, the metallic salt is either or both ofmagnesium nitrate and calcium nitrate. Claim 12 embodies the metallicsalt in Claim 6 and, therefore, the same effect as in Claim 6 isachieved.

According to Claim 13 of the present invention, there is provided abiosensor for measuring the concentration of a specific substance in asample solution, wherein organic acid or organic acid salt which has atleast one carboxyl group in its molecule is included in apreviously-provided reagent layer so that it is dissolved in the samplesolution and specifically reacts to the specific substance in the samplesolution. Therefore, it is possible to suppress an increase in abackground electric current with time, and suppress a needles reactionwith various contaminants existing in the blood, without preventing anenzyme reaction or the like, thereby providing a high performancebiosensor which is excellent in linearity (having a big slope and asmall intercept of a regression expression) and has small variationsamong individual sensors.

According to Claim 14 of the present invention, in the biosensor asdefined in Claim 13, the concentration of the specific substance ismeasured employing electrodes comprising at least a working electrodeand a counter electrode provided on an insulating support. Therefore, itis possible to provide a biosensor which is suitable for an examinationemploying electrodes.

According to Claim 15 of the present invention, in the biosensor asdefined in Claim 14, the reagent layer is formed on the electrodes or sothat the electrodes are arranged in a diffusion area where a reagent inthe reagent layer is dissolved and diffused in the sample solution, andthe reagent layer includes at least an enzyme and an electron transferagent. Therefore, it is possible to provide a biosensor which issuitable for an examination employing a reaction between an enzyme andan electron transfer agent.

According to Claim 16 of the present invention, in the biosensor asdefined in any of Claims 13 to 15, the organic acid is aliphaticcarboxylic acid, carbocyclic carboxylic acid, or heterocyclic carboxylicacid, or a substitution product or derivative of the organic acid. Claim19 embodies the organic acid in Claim 13 and, therefore, the same effectas in Claim 13 is achieved.

According to Claim 17 of the present invention, in the biosensor asdefined in any of Claims 13 to 15, the carboxylic acid is one ofglutaric acid, adipic acid, phthalic acid, and benzoic acid, or acombination of these acids. Claim 17 embodies the carboxylic acid inClaim 13 and, therefore, the same effect as in Claim 13 is achieved.

According to Claim 18 of the present invention, there is provided abiosensor for measuring the concentration of a specific substance in asample solution, wherein organic acid or organic acid salt which has atleast one carboxyl group and one amino group in its molecule is includedin a previously-provided reagent layer so that it is dissolved in thesample solution and specifically reacts to the specific substance in thesample solution. Therefore, it is possible to form the reagent layerclosely and homogeneously packed, thereby providing a biosensor whichcan dramatically enhance a response (sensitivity, linearity) of thesensor to the substrate concentration.

According to Claim 19 of the present invention, in the biosensor asdefined in Claim 18, the concentration of the specific substance ismeasured employing electrodes comprising at least a working electrodeand a counter electrode provided on an insulating support. Therefore, itis possible to provide a biosensor which is suitable for an examinationemploying electrodes.

According to Claim 20 of the present invention, in the biosensor asdefined in Claim 19, the reagent layer is formed on the electrodes or sothat the electrodes are arranged in a diffusion area where a reagent inthe reagent layer is dissolved and diffused in the sample solution, andthe reagent layer includes at least an enzyme and an electron transferagent. Therefore, it is possible to provide a biosensor which issuitable for an examination employing a reaction between enzyme and anelectron transfer agent.

According to Claim 21 of the present invention, in the biosensor asdefined in any of Claims 18 to 20, the organic acid is amino acid, or asubstitution product or derivative of the amino acid. Claim 21 embodiesthe organic acid in Claim 18 and, therefore, the same effect as in Claim18 is achieved.

According to Claim 22 of the present invention, in the biosensor asdefined in any of Claims 18 to 20, the amino acid is one of glycine,serine, proline, threonine, lysine, and taurine, or a combination ofthese acids. Claim 22 embodies the amino acid in Claim 18 and,therefore, the same effect as in Claim 18 is achieved.

According to Claim 23 of the present invention, there is provided abiosensor for measuring the concentration of a specific substance in asample solution, wherein a combination of at least two of sugar alcohol,metallic salt, organic acid or organic acid salt which has at least onecarboxyl group in its molecule, and organic acid or organic acid saltwhich has at least one carboxyl group and one amino group in itsmolecule, is included in a previously-provided reagent layer so that itis dissolved in the sample solution and specifically reacts to thespecific substance in the sample solution. Therefore, it is possible toprovide a highly-accurate biosensor which is excellent in stability andhas high response (sensitivity, linearity) of the sensor to thesubstrate concentration.

According to Claim 24 of the present invention, in the biosensor asdefined in any of Claims 1 to 23, the reagent layer further includes ahydrophilic polymer. Since the reagent layer includes the hydrophilicpolymer, formation of homogeneous reagent on the surface of theelectrodes is facilitated, and adhesion between the electrodes and thereagent is enhanced. Further, since the respective substances arehomogeneously dispersed in the reagent layer, homogeneous reagentformation can be realized, thereby providing a high performancebiosensor which has small variations among individual sensors.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating sensor response characteristics whenlactitol is added as sugar alcohol into a reagent solution, in a firstembodiment.

FIG. 2 is a diagram illustrating sensor response characteristics whenmaltitol is added as sugar alcohol into the reagent solution, in thefirst embodiment.

FIG. 3 is a diagram illustrating an increase in the background electriccurrent under a harsh environment when purified water is employed as asample solution in the first embodiment.

FIG. 4 is a diagram illustrating an increase in the whole blood responsevalue under a harsh environment when the whole blood is employed as thesample solution in the first embodiment.

FIG. 5 is a diagram illustrating an increase in the background electriccurrent under a harsh environment when purified water is employed as asample solution in a second embodiment.

FIG. 6 is a diagram illustrating an increase in the whole blood responsevalue under a harsh environment when the whole blood is employed as thesample solution in the second embodiment.

FIG. 7 is a diagram illustrating an increase in the background electriccurrent under a harsh environment when purified water is employed as asample solution in a third embodiment.

FIG. 8 is a diagram illustrating an increase in the background electriccurrent under a harsh environment when purified water is employed as asample solution in a fourth embodiment.

FIG. 9 is a diagram illustrating the whole blood response value when thewhole blood is employed as a sample solution in a fifth embodiment.

FIG. 10 is a diagram illustrating the whole blood response value whenthe whole blood is employed as the sample solution in the fifthembodiment.

FIG. 11 exemplifies an exploded perspective view of athree-electrode-system biosensor.

FIG. 12 exemplifies an exploded perspective view of atwo-electrode-system biosensor.

BEST MODE TO EXECUTE THE INVENTION Embodiment 1

Hereinafter, a biosensor according to a first embodiment of the presentinvention will be described. In the respective embodiments of theinvention described below, an enzyme sensor, which employs an enzyme asa molecular recognition element that specifically reacts to a specificsubstance in a sample solution, is exemplified.

FIG. 11 exemplifies an exploded perspective view of athree-electrode-system biosensor, and FIG. 12 exemplifies an explodedperspective view of a two-electrode-system biosensor. In FIGS. 11 and12, numeral 1 or 101 denotes an insulating support, on which a workingelectrode 2 or 102 and a counter electrode 3 or 103 are formed atprescribed positions in prescribed shapes. Further, in thethree-electrode-system biosensor shown in FIG. 11, a detecting electrode4 is formed on the insulating support 1 at a prescribed position in aprescribed shape. The detecting electrode 4 here serves as an electrodefor detecting shortage of a specimen quantity and, further, it can beemployed as a part of a reference electrode or the counter electrode.

Preferably, a material of the insulating support 1 or 101 ispolyethylene terephthalate, polycarbonate, polyimide, or the like.

Further, a conductive substance constituting each electrode is a simplesubstance such as a carbon or a noble metal such as gold, platinum,palladium, or the like, or a compound material such as a carbon paste ora noble-metal paste.

The simple substance such as carbon or noble metal such as gold,platinum, palladium, or the like can be easily formed into a conductivelayer on the insulating support 1 or 101 employing spattering depositionor the like, and the compound material such as a carbon paste or anoble-metal paste can be easily formed into a conductive layer on theinsulating support 1 or 101 employing screen printing or the like.

In the formation of the respective electrodes, the conductive layer isformed over the entire or partial insulating support 1 or 101 by theabove-mentioned spattering deposition or screen printing and,thereafter, slits are formed using a laser or the like to divide theconductive layer into the respective electrodes. Further, the electrodescan be similarly formed by screen printing or spattering depositionemploying a printing plate or a mask plate on which an electrode patternis previously formed.

A reagent layer 5 or 105, which includes an enzyme, an electron transferagent, a hydrophilic polymer, and sugar alcohol, is formed on theelectrodes formed as described above.

The first embodiment of the invention is characterized by that the sugaralcohol is included in the reagent layer 5 or 105, and the sugar alcoholprevents that the oxidized-form electron transfer agent gets contactwith a part of a reactive functional group existing in enzyme protein orhydrophilic polymer included in the reagent, so that the electrontransfer agent is denatured (reduced) from the oxidized-form toreduced-form in the reagent layer 5 or 105 formed on the electrodes.

Accordingly, in the biosensor with the above-described reagentformulation, under the environment where heat and moisture exist,particularly under the environment of high temperature and humiditywhere the temperature is over 30° C. and the humidity is over 80%, it ispossible to suppress a background electric current (noise electriccurrent) which occurs due to a reduction reaction between the electrontransfer agent and a part of enzyme protein or hydrophilic polymerincluded in the reagent layer 5 or 105, and is increased with time,thereby preventing the performance of the biosensor from beingdeteriorated.

Further, since the sugar alcohol is included in the reagent layer, aneedles reaction with various contaminants existing in the blood,especially in blood corpuscles, can be also suppressed, therebyproviding high performance biosensors which are excellent in linearity(having a big slope and a small segment of a regression expression) andhave less variations among the individual sensors.

The sugar alcohol included in the reagent layer 5 or 105 may be chainpolyhydric alcohol or cyclic sugar alcohol such as sorbitol, maltitol,xylitol, mannitol, lactitol, reduced paratinose, arabinitol, glycerol,ribitol, galactitol, sedoheptitol, perseitol, boremitol, styratitol,polygalitol, iditol, talitol, allitol, ishylitol, reduced starchsaccharified material, and ishylitol.

The same effect as above can also be achieved by using the stereoisomer,substitution product, or derivative of any of the sugar alcohols.

Among these sugar alcohols, maltitol and lactitol are the most suitablematerial since they are relatively low in the unit price, are easilyavailable, and are highly effective in suppressing the backgroundelectric current.

The addition quantity of the sugar alcohol is preferably 0.1-500 mM asthe reagent solution concentration, and more preferably, 1-100 mM.

Thereafter, in the biosensor as shown in FIG. 11 or 12, the spacer 6 or106 having the cutout part 7 or 107 and the cover 8 or 108 are attachedonto the reagent layer 5 or 105 and the electrodes 2, 3, 4, 102, 103,whereby a cavity through which the sample solution is supplied isformed.

A material suitable for the spacer 6 or 106 and the cover 8 or 108 maybe polyethylene terephthalate, polycarbonate, polyimide, polybutyleneterephthalate, polyamide, polyvinyl chloride, polyvinylidene chloride,nylon, and the like.

The supply of the sample solution to the biosensor having such cavity isrealized by capillary phenomenon, and an air vent 9 or 109 for lettingair out of the biosensor is required in the cavity for smooth supply ofthe sample solution.

The air vent 9 or 109 can be arranged at any position in the cavityunless it prevents the supply of the sample solution.

In the so-formed biosensor, the electric current value obtained by thereaction between a specific component in the sample solution and thereagent layer 5 or 105 including an enzyme or the like is read by anexternal measuring device connected through lead parts 10, 11, 12, 110,111 of the working electrodes 2 or 102, the counter electrode 3 or 103,and the detecting electrode 4.

Embodiment 2

Hereinafter, a biosensor according to a second embodiment of the presentinvention will be described.

The biosensor according to the second embodiment of the invention is thebiosensor shown in FIG. 11 or 12 whose reagent layer 5 or 105 is formedof an enzyme, an electron transfer agent, a hydrophilic polymer, and ametallic salt. Other constituents are the same as those of theabove-described biosensor according to the first embodiment and,therefore, descriptions thereof will be omitted.

The second embodiment of the invention is characterized by that ametallic salt is included in the reagent layer 5 or 105, and themetallic salt prevents that the oxidized-form electron transfer agentgets contact with a part of a reactive functional group existing inenzyme protein and hydrophilic polymer included in the reagent, so thatthe electron transfer agent is denatured (reduced) from the oxidizedform to reduced form in the reagent layer 5 or 105 formed on theelectrodes.

Accordingly, in the biosensor with the above-described reagentconstitution, under the environment where heat and moisture exist,particularly under the environment of high temperature and humiditywhere the temperature is over 30° C. and the humidity is over 80%, it ispossible to suppress a background electric current (noise electriccurrent) which occurs due to the reduction reaction between the electrontransfer agent and a part of enzyme protein or hydrophilic polymer andis increased with time, thereby preventing the performance of thebiosensor from being deteriorated.

As the metallic salt included in the reagent layer 5 or 105, metallicsalt sulfate or metallic salt nitrate is particularly effective. Asmetallic salt sulfate, metallic salt hydrogensulfate, metallic saltsulfite, metallic salt hydrogensulfite, or metallic salt hyposulfite,for example, there are aluminium sulfate, magnesium sulfate, zincsulfate, antimony sulfate, indium sulfate, uranyl sulfate, uraniumsulfate, cadmium sulfate, potassium sulfate, gallium sulfate, calciumsulfate, silver sulfate, chromium sulfate, cobalt sulfate, potassiumbisulfate, zirconium sulfate, mercury sulfate, tin sulfate, strontiumsulfate, caesium sulfate, cerium sulfate, thallium sulfate, titaniumsulfate, iron sulfate, copper sulfate, sodium sulfate, lead sulfate,nickel sulfate, neodymium sulfate, vanadium sulfate, palladium sulfate,barium sulfate, bismuth sulfate, praseodymium sulfate, berylliumsulfate, manganese sulfate, lanthanum sulfate, lithium sulfate, rubidiumsulfate, aluminium potassium sulfate, aluminium sodium sulfate, uranylpotassium sulfate, potassium chromium sulfate, disodium magnesiumsulfate, magnesium dipotassium sulfate, manganese caesium sulfate,rubidium aluminium sulfate, potassium hydrogensulfate, sodiumhydrogensulfate, potassium sulfite, calcium sulfite, sodium sulfite,barium sulfite, bismuth sulfite, sodium hyposulfite, potassiumbisulfite, sodium bisulfite, and the like, while, as metallic saltnitrate, metallic salt hydrogennitrate, metallic salt nitrite, metallicsalt hydrogennitrite, or metallic salt hyponitrite, there are aluminiumnitrate, magnesium nitrate, zinc nitrate, antimony sulfate, ytterbiumnitrate, yttrium nitrate, indium nitrate, uranyl nitrate, erbiumnitrate, cadmium nitrate, gadolinium nitrate, potassium nitrate, calciumnitrate, silver nitrate, chromium nitrate, cobalt nitrate, samariumnitrate, zirconium nitrate, dysprosium nitrate, mercury nitrate, tinnitrate, strontium nitrate, caesium nitrate, cerium nitrate, thalliumnitrate, iron nitrate, terbium nitrate, copper nitrate, thorium nitrate,sodium nitrate, lead nitrate, nickel nitrate, neodymium nitrate,palladium nitrate, barium nitrate, bismuth nitrate, praseodymiumnitrate, beryllium nitrate, holmium nitrate, manganese nitrate, europiumnitrate, lanthanum nitrate, lithium nitrate, ruthenium nitrate, rubidiumnitrate, rhodium nitrate, thallium mercury nitrate, potassium nitrite,silver nitrite, calcium nitrite, sodium nitrite, potassiumcobaltinitrite, sodium cobaltinitrite, sodium hyponitrite, and the like.

Among these metallic salts, magnesium sulfate, calcium sulfate,magnesium nitrate, and calcium nitrate are particularly suitable becausethey are highly effective in preventing moisture absorption.

The addition quantity of the metallic salt is suitably 0.1-500 mM as areagent solution concentration, and more suitably, 1-50 mM.

Embodiment 3

Hereinafter, a biosensor according to a third embodiment of the presentinvention will be described.

The biosensor according to the third embodiment of the invention has areagent layer 5 or 105 shown in FIG. 11 or 12, which is formed of anenzyme, an electron transfer agent, a hydrophilic polymer, and anorganic acid or organic acid salt which has at least one carboxyl groupin a molecule. Other constituents are the same as those of theabove-described biosensor according to the first embodiment and,therefore, descriptions thereof will be omitted.

The third embodiment of the invention is characterized by that anorganic acid or organic acid salt having at least one carboxyl group ina molecule is included in the reagent layer 5 or 105, and the organicacid or organic acid salt prevents that the oxidized-form electrontransfer agent gets contact with a part of a reactive functional groupexisting in enzyme protein and hydrophilic polymer included in thereagent, so that the electron transfer agent gets denatured (reduced)from the oxidized form to reduced form in the reagent layer 5 or 105formed on the electrodes.

Accordingly, in the biosensor with the above-described reagentconstitution, under the environment where heat and moisture exist,particularly under the environment of high temperature and humiditywhere the temperature is over 30° C. and the humidity is over 80%, it ispossible to suppress a background electric current (noise electriccurrent) which occurs due to the reduction reaction between a part ofenzyme protein or hydrophilic polymer and the electron transfer agentand is increased with time, thereby preventing the performance of thebiosensor from being deteriorated.

Further, since the organic acid or organic acid salt having at least onecarboxyl group in a molecule is included in the reagent layer, a needlesreaction with various contaminants existing in the blood, particularlyin blood corpuscles, can be also suppressed, thereby providing highperformance biosensors which are excellent in linearity (having a bigslope and a small intersept of a regression expression) and have lessvariations among individual sensors.

The organic acid or organic acid salt which has at least one carboxylgroup in a molecule and is included in the reagent layer 5 or 105 may bealiphatic carboxylic acid, carbocyclic carboxylic acid, heterocycliccarboxylic acid, or their salt.

For example, the aliphatic carboxylic acid may be malonic acid, succinicacid, glutaric acid, adipic acid, maleic acid, fumaric acid, and theirsalt.

The degree of the effect becomes larger as the straight chain is longerand the molecular weight is larger, and particularly, an organic acid ororganic acid salt having three or more hydrocarbon chains is desirable.Further, as for a reagent employed for the biosensor, a reagent havingmore hydrophilic functional groups in the molecular structure ispreferable since the reagent is required to be highly soluble in water.

Further, the carbocyclic carboxylic acid may be benzoic acid, phthalicacid, isophthalic acid, terephthalic acid, and their salt, and the sameeffect as the above can be obtained by employing these.

The heterocyclic carboxylic acid may be 2-phthalic-acid, nicotinic acid,isonicotinic acid, and their salt, and the same effect as the above canbe obtained by employing these.

Further, in addition to the above-described aliphatic and carbocycliccarboxylic acid, as well as heterocyclic carboxylic acid or carboxylatesalt, malic acid, oxaloacetic acid, citric acid, ketoglutaric acid, andtheir salt, for example, in which functional groups of the carboxylicacid or carboxylate salt are partially replaced by other functionalgroups, can also achieve the same effect as described above.

Among such organic acids or organic acid salts, glutaric acid, adipicacid, phthalic acid, and benzoic acid are most suitable.

The addition quantity of the organic acid or organic acid salt issuitably 0.01-100 mM as a reagent solution concentration and, moresuitably, 0.1-10 mM.

Embodiment 4

Hereinafter, a biosensor according to a fourth embodiment of the presentinvention will be described.

The biosensor according to the fourth embodiment of the invention has areagent layer 5 or 105 shown in FIG. 11 or 12, which is formed of anenzyme, an electron transfer agent, a hydrophilic polymer, and anorganic acid or organic acid salt which has at least one carboxyl groupand one amino group in a molecule. Other constituents are the same asthose of the above-described biosensor according to the first embodimentand, therefore, descriptions thereof will be omitted.

The fourth embodiment of the invention is characterized by the anorganic acid or organic acid salt having at least one carboxyl group andone amino group in a molecule is included in the reagent layer 5 or 105,and addition of the organic acid or organic acid salt into the reagentlayer 5 or 105 provides the effect that the surface state of the reagentlayer 5 or 105 can be made extremely smooth and homogeneous. Althoughthe reagent layer 5 or 105 is easily crystallized in the process ofdrying the reagent solution when inorganic salt such as potassiumferricyanide employed as an electron transfer agent is included in thereagent layer, the crystallization of the inorganic salt can beprevented by the organic acid or organic acid salt which has at leastone carboxyl group and one amino group in a molecule and is included inthe reagent.

Since the inorganic salt prevented from being crystallized exists in thereagent layer as a particulate, it can get close and uniform contactwith an enzyme molecule, resulting in a reagent layer condition which isexcellent in efficiency of electron transfer with the enzyme molecule.

Further, since the solubility of the reagent layer can be enhanced, thesensitivity and linearity of the sensor can be dramatically improved.

The organic acid or organic acid salt, which has at least one carboxylgroup and one amino group in a molecule and is included in the reagentlayer 5 or 105, may be an organic acid or organic acid salt such asglycine, alanine, valine, leucine, isoleucine, serine, threonine,methionine, asparagine, glutamine, arginine, lysine, histidine,phenylalanine, tryptophan, proline, or their salt, or sarcosine,betaine, taurine, or the like.

The same effect as above can be achieved by employing the substitutionproduct or derivative of any of these organic acids or organic acidsalts.

Among the organic acids or organic acid salts, glycine, serine, proline,threonine, lysine, and taurine are particularly suitable since they areeffective in preventing crystallization.

The addition quantity of the organic acid or organic acid salt issuitably 0.1-1000 mM as a reagent solution concentration and, moresuitably, 10-500 mM.

In the first to fourth embodiments of the invention, descriptions aregiven of the cases where sugar alcohol, metallic salt, organic acid ororganic acid salt which has at least one carboxyl group in a molecule,and organic acid or organic acid salt which has at least one carboxylgroup and one amino group in a molecule are respectively added to thereagent layer 5 or 105, these additives may be appropriately combined.

The enzyme, which is included in the reagent in the first to fourthembodiments of the invention, may be glucose oxidase, lactate oxidase,cholesterol oxidase, cholesterol esterase, uricase, ascorbia acidoxidase, bilirubin oxidase, glucose dehydrogenase, lactatedehydrogenase, or the like, and the electron transfer agent may bepotassium ferricyanide, p-benzoquinone or its derivative, phenazinemethosulfate, methylene blue, ferrocene or its derivative, or the like.

In the first to fourth embodiments of the invention, the reagent layer 5or 105 includes a hydrophilic polymer, whereby the sample solution has aviscosity, and the formation of the reagent on the electrodes isfacilitated and homogenized and, further, the adhesion between theelectrodes and the reagent is enhanced. Furthermore, the hydrophilicpolymer included in the reagent layer makes the condition of the reagentcrystal after the reagent is dried even and homogeneous, resulting in ahigh-accurate biosensor.

Hydrophilic polymers used for the above-described purpose may becarboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropylcellulose,methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose,carboxymethyl ethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone,polyamino acid such as polylysine, polystyrene sulfonate, or gelatineand its derivative, acrylic acid and its salt, methacrylic acid and itssalt, starch and its derivative, maleic anhydride and its salt, andagarose gel and its derivative.

While in the first to fourth embodiments of the invention theabove-described reagent layer 5 or 105 is provided on the electrodes,specifically the reagent layer 5 or 105 can be arranged over the entiresurface or part of the electrodes. Besides, the reagent layer 5 or 105may be arranged within a range where the performance of the biosensor isnot deteriorated, i.e., a range where the electrodes are provided withinan area in which the reagent in the reagent layer is dissolved anddiffused into the sample solution.

Example 1

A palladium thin film of approximately 10 nm thick is formed over theentire surface of an insulating support comprising polyethyleneterephthalate by spattering deposition and, thereafter, slits are formedin part of the thin film using a YAG laser so as to divide the thin filminto a working electrode, a counter electrode, and a detectingelectrode. On the electrodes, an aqueous solution including an enzyme(glucose oxidase), an electron transfer agent (potassium ferricyanide),a hydrophilic polymer (carboxymethyl cellulose), and sugar alcohol iscircularly dropped centering around the working electrode so as to coverparts of the counter electrode and the detecting electrode, and then itis dried, thereby forming a reagent layer. On the reagent layer, aspacer having a cutout part and comprising polyethylene terephthalateand a cover having an air vent and comprising polyethylene terephthalateare attached, thereby manufacturing a three-electrode-system sensor formeasuring a blood sugar level, in which a cavity as a capillary forleading blood is formed.

FIG. 1 illustrates sensor response characteristics when lactitol isadded into the reagent solution as sugar alcohol, in the case where thesample solution is whole blood and the lactitol concentration is variedin four levels. Likewise, FIG. 2 illustrates sensor responsecharacteristics in the case where maltitol is added as sugar alcohol andthe maltitol concentration is varied in four levels. Here, a sensor inwhich the sugar alcohol addition concentration (the concentration as asample aqueous solution) is 0 is handled as a conventional sensor, andsensors in which the sugar alcohol addition concentrations are 5, 10,25, and 50 mM are employed as invention sensors.

FIG. 3 illustrates change of a background electric current value withtime under a harsh environment (exposure in temperature of 30° C. andhumidity of 80%), which is measured using the sensor manufactured asdescribed above. Purified water including no glucose is employed as asample solution. FIG. 4 illustrates change of a sensor response valuewith time in the case where whole blood adjusted to have the glucoseconcentration of 80 mg/dL is employed as a sample solution. In eithercase, there are four points of measurement in total: just after thesensor is manufactured (0-hour), 6-hour, 12-hour, and 24-hour after themanufacture.

The current measurement condition is as follows. After confirming thatthe cavity is filled with the sample solution, enzyme reaction ispromoted for twenty-five seconds. Thereafter, a voltage of 0.2V isapplied among the working electrode, the counter electrode, and thedetecting electrode, and an electric current value obtained five secondsafter the application is measured. Here, the detecting electrode is alsoemployed as a part of the counter electrode.

The number of measurement n is n=10 for each concentration andmeasurement time, and the average thereof is plotted in the figures.

As is evident from FIG. 1, the response characteristics of the sensor inwhich lactitol is added as sugar alcohol tend to be high particularly ina range where the glucose concentration is high, i.e., over 400 mg/dL,as compared with the conventional sensor including no sugar alcohol, andthis result indicates that excellent response characteristics with afavorable regression expression (having a small intercept and a bigslope) are obtained.

As is evident from FIG. 2, also when maltitol is employed as sugaralcohol, excellent response characteristics are obtained as in theabove-mentioned case of employing lactitol.

Furthermore, as is evident from FIGS. 3 and 4, in the sensor to whichthe sugar alcohol is added, increase in the background electric currentin the exposure environment under the high temperature and humidity isefficiently suppressed, resulting in excellent preservation stabilitywith small variations of the sensor response value with time.

Example 2

A sensor for measuring a blood sugar level is manufactured by the sameprocedure as in the first example. In this second example, magnesiumsulfate as metallic salt sulfate is added instead of sugar alcohol as anaddition agent for suppressing increase of a background electric currentwith time.

FIG. 5 illustrates change of a background electric current value withtime under a harsh environment (exposure in temperature of 30° C. andhumidity of 80%), which is measured employing the sensor manufactured asdescribed above. Purified water including no glucose is employed as asample solution. FIG. 6 illustrates change of a sensor response valuewith time in the case where whole blood adjusted to have the glucoseconcentration of 80 mg/dL is employed as a sample solution.

In either case, there are four points of measurement in total: justafter the sensor is manufactured (0-hour), 6-hour, 12-hour, and 24-hourafter the manufacture.

The number of measurement n is the same as that in the first example.

As is evident from FIGS. 5 and 6, also in the sensor in which metallicsalt sulfate is added, as well as sugar alcohol in the first example,increase in the background electric current in the exposure environmentunder the high temperature and humidity is efficiently suppressed,resulting in excellent preservation stability with small variations ofthe sensor response value with time.

Example 3

An electrode layer comprising a working electrode and a counterelectrode is formed on an insulating support comprising polyethyleneterephthalate by screen printing, and a reagent layer including anenzyme (glucose oxidase), an electron transfer agent (potassiumferricyanide), a hydrophilic polymer (carboxymethyl cellulose), andaliphatic carboxylic acid (concentration of which is 5 mM as a samplesolution) is formed on the electrode layer. Thereafter, a spacercomprising polyethylene terephthalate and a cover also comprisingpolyethylene terephthalate are attached, thereby manufacturing atwo-electrode-system sensor for measuring a blood sugar level, in whicha cavity as a capillary for leading blood is formed.

Here, four kinds of two-electrode-system sensors in total aremanufactured: three sensors respectively including, as organic acids,malonic acid (HOOC—CH₂—COOH) as aliphatic carboxylic acid, glutaric acid(HOOC—CH₂—CH₂—CH₂—COOH), and adipic acid (HOOC—CH₂—CH₂—CH₂—CH₂—COOH),and a conventional sensor including no aliphatic carboxylic acid.

FIG. 7 illustrates background electric currents under a harshenvironment (temperature of 40° C. and humidity of 80%), which aremeasured employing the four sensors manufactured as mentioned above.Purified water including no glucose is employed as a sample solution.There are four points of measurement in total: just after the sensor ismanufactured (0-hour), 7-day, 14-day, and 30-day after the manufacture.The measurement condition is as follows. The cavity is filled with thesample solution (purified water), and the reaction is promoted fortwenty-five seconds. Thereafter, a voltage of 0.5V is applied betweenthe working electrode and the counter electrode, and an electric currentvalue obtained five seconds after the application is measured.

The number of measurement n is n=10 for each measurement point, and theaverage thereof is plotted in FIG. 7.

As is evident from FIG. 7, increase in the background electric currentis reliably suppressed in the sensors added with the aliphaticcarboxylic acids, and the pace of the increase is reduced in the orderof malonic acid, glutaric acid, and adipic acid, thereby suggesting thatthe sensor with more complicated molecular structure, longer straightchain, and larger molecular weight is more effective in suppressing theincrease in the background electric current. The electric current valuethus obtained corresponds to the quantity of potassium ferrocyanidewhich is generated by a reaction between glucose oxidase and potassiumferricyanide as well as carboxymethyl cellulose and potassiumferricyanide.

Example 4

A biosensor is manufactured by the same procedure as in the thirdexample and the same evaluation as in the third example is made. Thisfourth example employs three kinds of organic acids as follows: benzoicacid and phthalic acid as carbocyclic carboxylic acids, and malic acid(derivative of succinic acid) with the structure in which a part ofhydrocarbon chain of succinic acid is replaced by a hydroxyl group.

As is evident from FIG. 8, the effect of suppressing increase in thebackground electric current is confirmed whichever organic acid ofbenzoic acid, phthalic acid, or malic acid is employed as in the thirdexample.

Example 5

An electrode layer comprising a working electrode and a counterelectrode is formed on an insulating support comprising polyethyleneterephthalate by screen printing, and a reagent layer including anenzyme (glucose dehydrogenase with pyrrolo-quinoline quinone ascoenzyme), an electron transfer agent (potassium ferricyanide), ahydrophilic polymer (carboxymethyl cellulose), aliphatic carboxylic acid(phthalic acid), and amino acid is formed on the electrodes. Thereafter,a spacer comprising polyethylene terephthalate and a cover comprisingpolyethylene terephthalate as well are attached, thereby manufacturing atwo-electrode-system sensor for measuring a blood sugar level, in whicha cavity as a capillary for leading blood is formed.

In this fifth example, eight kinds of sensors in total are manufacturedas follows: sensors respectively including, as organic acids, glycine(Gly), serine (Ser), proline (Pro), threonine (Thr), lysine (Lys),sarcosine (derivative of glycine), and taurine, which are amino acidshaving at least one carboxyl group and one amino group in a molecule,and a conventional sensor including no amino acid.

FIGS. 9 and 10 illustrate sensor response characteristics when glucosein human whole blood is measured employing the eight sensorsmanufactured as described above. This fifth example employs the glucoseconcentrations in whole blood, 40, 80, 350, 600, and 700 mg/dl.

The measurement condition is as follows. After the cavity is filled withthe sample solution (human whole blood), a reaction is promoted fortwenty-five seconds. Thereafter, a voltage of 0.5V is applied betweenthe working electrode and the counter electrode, and an electric currentvalue obtained five seconds after the application is measured.

The number of measurement n is n=20 for each concentration, and theaverage thereof is plotted in the figure.

As is evident from FIGS. 9 and 10, dramatic enhancement of the responsevalue and linearity is confirmed particularly in a range where theglucose concentration is higher than 480 mg/dL, as compared with theconventional sensor including no amino acid, although there are slightdifferences in the response values according to the kinds of aminoacids.

Table 1 shows variations of the sensor response value at the measurementof n=20 by CV values. As is evident from table 1, considerableimprovement of the CV value is recognized in the sensors of theinvention which are added with amino acids. The reason is as follows.Since the amino acid added into the reagent layer prevents potassiumferricyanide from being crystallized, the reagent layer is formedsmoothly and homogeneously, whereby solubility and diffusion of thereagent becomes homogeneous, resulting in a reduction of responsevariations.

TABLE 1 Concentration (mg/dl) 40 80 350 600 700 Conventional 3.25% 2.48%2.05% 2.11% 4.15% sensor Gly-133 mM 2.15% 1.75% 1.55% 1.28% 1.05% Ser-95mM 2.54% 2.11% 1.75% 1.45% 1.22% Pro-87 mM 2.38% 1.98% 1.69% 1.40% 1.18%Thr-84 mM 2.18% 2.00% 1.65% 1.39% 1.24% Lys-HCl-55 mM 2.48% 1.89% 1.58%1.41% 1.14% Sarcosine- 2.65% 2.08% 1.68% 1.35% 1.25% 122 mM Taurine-80mM 2.18% 1.78% 1.43% 1.22% 1.10%

While the first to fifth examples describe the biosensors for measuringthe glucose concentration in blood, the sample solution and substance astargets of measurement and the type of biosensor are not restrictedthereto. For example, a liquid vital specimen such as saliva,intercellular substance liquid, urine, or sweat, as well as food ordrinking water, can also be employed as a target sample solution besidesblood. Further, lactic acid, cholesterol, uric acid, ascorbic acid, orbilirubin, can also be also employed as a target substance besidesglucose. Furthermore, As a current measurement method, there are thethree-electrode system constituted by the working electrode, the counterelectrode, and the detecting electrode, which is employed in the firstand second examples, or the two-electrode system constituted by theworking electrode and the counter electrode, which is employed in thethird and fifth examples, and the same effects as described above can beobtained whichever system is employed. The three-electrode systemenables more accurate measurement as compared with the two-electrodesystem.

While the examples are described exemplifying the enzyme sensor as abiosensor, the present invention is similarly applicable to a biosensorutilizing antibodies, microorganisms, DNA, RNA, or the like, besidesenzyme, as a molecular recognition element which specifically reacts toa specific substance in a sample solution.

APPLICABILITY IN INDUSTRY

A biosensor according to the present invention includes either one or acombination of sugar alcohol, metallic salt, organic acid or organicacid salt which has at least one carboxyl group in a molecule, ororganic acid or organic acid salt which has at least one carboxyl groupand amino group in a molecule in its reagent, so that increase of abackground electric current with time can be suppressed, a needlesreaction with various contaminants existing in the blood can besuppressed, or the reagent layer can be closely and homogeneouslyformed, without preventing an enzyme reaction or the like, therebyproviding a highly stable and efficient biosensor.

1. A biosensor for measuring the concentration of a specific substancein a sample solution, wherein taurine is included in apreviously-provided reagent layer, including electrodes comprising atleast a working electrode and a counter electrode which are composed ofan electric conductive substance, said electrodes being provided on aninsulating support, wherein the concentration of the specific substanceis measured employing said electrodes.
 2. The biosensor as defined inclaim 1, wherein the reagent layer is formed on the electrodes or sothat the electrodes are arranged in a diffusion area where a reagent inthe reagent layer is dissolved and diffused in the sample solution, andthe reagent layer includes at least an enzyme and an electron transferagent.
 3. The biosensor as defined in claim 1, wherein thepreviously-provided reagent layer also includes maltitol or lactitol. 4.The biosensor as defined in claim 3, wherein the reagent layer furtherincludes a hydrophilic polymer.
 5. The biosensor as defined in claim 1,wherein the reagent layer further includes a hydrophilic polymer.
 6. Thebiosensor as defined in claim 1, wherein taurine is included in thepreviously-provided reagent layer so that it is dissolved in the samplesolution.